PROJECTSUMMARY Our studies are focused on a regulatory network utilized by bacteria to effect an orderly progression of cell cycle events in a manner that is sensitive to environmental conditions and is capable of producing differentiated cell types. Our primary objective is to understand how a regulatory network directs cell cycle events in order to promote chronic host colonization. To this end, we use Sinorhizobium meliloti as a model systembecauseitcangrowinthesoilasafree-??livingbacteriumorcolonizetherootsofplantsasabeneficial symbiont to establish a chronic intracellular infection. S. meliloti undertake novel cell cycle modifications during symbiosis, however the underlying molecular mechanisms that direct these events are unknown. By executingtheaimsdescribedinthisproposal,wewilldevelopamechanisticmodelforcellcycleregulation.A recently identified two-??component signal transduction pathway is known to control cell cycle progression. CbrAisahistidinekinasethatfunctionsatthetopofthispathwaytoregulatetheactivityofCtrA,anessential DNA-??binding response regulator that controls the transcription of regulatory and effector proteins in a temporalfashionasthecellcycleprogresses.WeidentifieddivLasacomponentoftheCbrApathwayandwill further characterize its function. Using cell biological methods, we will determine its role in cell cycle regulationandsymbiosis.Wewillalsouseageneticsuppressorscreentoidentifycellularfactorsthatinteract willDivLandconfirmtheseinteractionsthroughinvitrobiochemistryandinvivoproteinlocalizationstudies. We identified MorA as a cell cycle regulator that is functionally redundant with CbrA but under distinct growth conditions. We will determine how MorA is integrated into the two-??component pathway using biochemical assays to identify its cognate response regulator(s). In addition, we will examine how MorA activity is limited to certain environmental conditions through a screen for regulatory factors. In the process, our studies will generate genetic tools required to probe the function of a pathway that includes several proteinswhoseactivityisessentialtoviabilityandthereforechallengingtostudyonafunctionallevelinvivo. Ultimately, we will develop a mechanistic model for cell cycle regulation in the experimentally tractable S. melilotithatwillprovideabasisfordissectingcellcyclecontrolsinrelatedpathogenicbacteria.Ourresearchis therefore of broad importance to understanding both cell cycle progression and diverse host-??microbe interactions.